Plant Physiol. (1980) 65, 995-10030032-0889/80/65/0995/09/$00.50/0

Slow Adaptive Changes in Urease Levels of Tobacco CellsCultured on Urea and Other Nitrogen Sources'

Received for publication August 16, 1979 and in revised form November 16, 1979

THOMAS A. SKOKUT2 AND PHILIP FILNERMSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

Tobacco (cv. Xanthi) XD cells cultured for more than a year on urea asthe sole source of nitrogen have urease activities about four times higherthan cels which have been cultured on nitrate. When ceUs which hadalways been grown on nitrate were transferred to urea, the urease activityin these cells remained at a lower level for eight transfers (40 generations),then gradually increased 4-fold during the next seven to 10 transfers. CeUswith high urease activity multiplied 19% more rapidly and accumulated lessurea than cells with low urease activity. These findings suggest thatelevated urease accelerates urea assimilation; therefore, urea limitedgrowth. Clones of ceUs with low urease activity responded in the same wayas uncloned populations when transferred from nitrate to urea, indicatingthat high urease ceUs originate from low urease cells, rather than from apreexisting subpopulation of high urease ceUs. The urease levels in clonesof cells from a population with high urease activity were three to seventimes the low urease level. The observed dependence of urease activity ongenerations of growth on urea was matched with a model in which highurease cells originated at mitosis of low urease ceUls at a frequency of 8x 10', then multiplied 19% more rapidly than low urease cells. Thisfrequency is about 103 greater than that of other biochemical variantspreviously isolated from XD cells. The high urease activity graduallydeclined in cels transferred from urea to other nitrogen sources, but roserapidly when such ceUls were returned to urea, indicating the existencewithin the cels of some form of record of their ancestors' growth on urea.The data indicate the existence of a mechanism for generation, at unusuallyhigh frequency, of metastable variants with high urease activity. Thismechanism, coupled with enrichment for the variants' progeny by virtue oftheir higher multiplication rate on urea, can account for the observed slowincrease in urease activity of the population. It is suggested that themolecular basis of the urease increase may be gene amplification, based onanimal cell models. An alternative hypothesis, namely a specific responseinduced in all cells by urea and manifested as a very slow adaptive increasein urease, has not been ruled out.

Urea assimilation can be accomplished by two different enzymicpathways. One pathway involves the direct hydrolysis of urea toNH4' and C02, catalyzed by urease. The other pathway involvescarboxylation of urea to form allophanate, followed by hydrolysisof allophanate to NH4' and CO2 (23). To date, the urea carbox-ylase/allophanate hydrolase system has been found in fungi (24)

'This work was supported by the United States Department of Energyunder contract EY-76-C-02-1338. This work is taken from a dissertationsubmitted to Michigan State University by T. A. S. in partial fulfillmentof the requirements for degree of Ph.D. in botany.

Present address: Biology Department, Washington University, St.Louis, Missouri 63130.

and algae (26), but not in higher plants (26). On the other hand,urease activity has been detected in the tissues ofnumerous speciesof higher plants (3, 5, 9, 17, 20, 29). Therefore, it appears that theonly mechanism by which higher plants utilize urea is hydrolysisby urease.

Increases in urease activity in response to urea have beenreported to occur in potato plants (18), rice plants (17), duckweed(5), and cell cultures of soybean (20). These increases occurredwithin a few hours to a few days after beginning the exposure tourea. We report different results of a study of urease activity inthe XD line of cultured tobacco cells. In these cells, urease activityincreases only after months of cell multiplication in mediumcontaining urea as the sole source of nitrogen. A preliminaryaccount of this work has appeared (25).

MATERIALS AND METHODS

Growth Media. Medium M- ID, which contains 2.5 mm nitrate,and nitrate-free M- ID, in which equimolar amounts of the chlo-rides of K and Ca are substituted for the corresponding nitrates,were prepared as described previously (6, 7). Ammonium succi-nate M-ID was prepared by the addition of 3 mm ammoniumchloride and 1.5 mm succinic acid to nitrate-free M- ID. Casaminoacids M- ID was prepared by addition of 0.1% w/v casamino acids(Difco Laboratories, Detroit) to nitrate-free M-ID. Urea M-IDwas prepared by addition of 0.3% (v/v) 1 M urea in water tonitrate-free M- ID. All media were autoclaved for 20 min, and theurea and casamino acids stock solutions were sterilized by filtra-tion through a sterile membrane filter (type HA, 0.22 Am pore,Millipore Corp. The urea used was obtained from two sources.Urea from Fisher Scientific Co. was first purified by batchwiseadsorption of anions on Bio-Rad AG 2-X8 ion exchange resin inthe hydroxyl form. Urea (ultrapure grade) obtained fromSchwarz/Mann was used without further purification. All media,and the urea and casamino acids stock solutions were adjustedwith NaOH to pH 6.2 prior to sterilization.Growth of Cells. Cells of tobacco (Nicotiana tabacum L. cv.

Xanthi, line XD) were grown on the various liquid media de-scribed above under the culture conditions described previously(6). Cultures were maintained by inoculating 500-ml portions ofmedia with 25-ml aliquots of 12- to 14-day-old stationary phasecultures of cells grown on nitrate or of 14- to 18-day old stationaryphase cultures of cells grown (more slowly) on urea. The freshweights of inocula were 0.5-1 g/liter.

Harvesting of Cells and Preparation of Extracts. Cells wereharvested by vacuum filtration on Whatman No. I filter paperand rinsed with deionized distilled H20. After determining thefresh weight, the cells were suspended in ice-cold 50 mm Bicine3-NaOH (Sigma) (pH 7.2) containing 5 mm dithioerythritol (Sigma).Five ml buffer g-1 fresh weight were used. The cells were homog-

'Abbreviation: Bicine: N,N'-bis(2-hydroxyethyl)glycine.995

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SKOKUT AND FILNER

enized by 30 strokes of a motor-driven Teflon glass homogenizer(A. H. Thomas Co., Philadelphia, Pa.) at 4 C and then sedimentedat 10,000 rpm for 20 min in a Sorvall refrigerated centrifuge. Thesupernatant was then centrifuged at 100,000g for 1 h at 4 C in aBeckman ultracentrifuge using a type 65 rotor. The resultingsupernatant fraction was used as a crude enzyme preparation. Itwas assayed after appropriate dilution with the homogenizingbuffer. Occasionally, this supernatant fraction was dialyzedagainst 2 liters 50 mm Bicine-NaOH buffer (pH 7.2) with 0.5 mmdithioerythritol for 4-5 h, to remove ammonium ions.Assay for Urease. Urease activity was determined by measuring

the release of ammonium ions when urea was incubated with thecell extract. The complete reaction mixture contained: 50 mmBicine-NaOH (pH 7.2); 5 mm dithioerythritol; 50 mm urea; andfrom 1 to 600 ,ug protein of enzyme extract. For each assay, I mlof the reaction mixture was prepared at ice temperature in a 10-ml test tube. The urea was the last addition to the tube. Thereaction was started by placing the tubes in a water bath at 30 C.Incubation times were 0 and 60 min. The reaction was terminatedby adding 0.5 ml of ZnSO4, 50 g liter-', followed by 0.5 ml of 0.2M Ba(OH)2 (29). The precipitate formed was sedimented by cen-trifugation at 1,000 rpm for 5 min, and 1 ml of the resultingsupernatant solution was used for determination of ammoniumion concentration by the phenol-hypochlorite method (proceduredescribed below). Zero time assays were used as controls. Theincrease in ammonium ion concentration in the reaction mixturefrom 0 to 60 min was taken as the measure of urease activity. Dataare expressed in [imol NH4' released h-' g-' fresh weight or mg-'protein. All assays were performed in duplicate. Reaction mixtureswhich were heated in a boiling water bath for 5 min prior to the60-min incubation at 30 C exhibited no increase in ammonium.The components of the reaction mixture, cell extract and precipi-tating agents did not interfere with the ammonium assay. Whena cell extract was supplemented with a known concentration ofNH4Cl and was then subjected to the entire assay procedureomitting the 60-min incubation, the value obtained for the am-monium concentration (minus the amount originally found in theextract) agreed to within ±5% with that obtained with a standardof the same concentration dissolved in H20.Assay for Ammonium Ion. Ammonium ion concentration was

determined by a procedure similar to that of Kaplan (13). Thereaction was initiated by adding successively to a l-ml sample, 5ml 0.2 mm sodium nitroprusside in 1% (w/v) phenol and 5 ml0.125 N NaOH in 0.05% (v/v) NaOCl. After being mixed on aVortex stirrer, the solutions were incubated at room temperaturefor 30 min during which time a blue color developed. A at 625 nmwas then determined. The A of a blank which consisted of distilledH20 plus the above reagents was subtracted from all values. Theconcentrations of ammonium in the unknown samples were cal-culated from a standard A curve obtained with known concentra-tions of NH4CL.

Determination of Urea. The concentration of urea in cell ex-tracts was determined by first hydrolyzing it to NH4' and CO2using a commercially available urease and subsequently assayingfor ammonium ions using the procedure described above. Cellextract (0.5 ml) was mixed with 0.5 ml urease (1 mg/ml, purifiedfrom jack bean seeds with a specific activity 3,200 units/g; Sigma)and incubated at 30 C for 60 min. The reaction was stopped withthe same precipitating agents used in the assay for urease. Am-monium ion assays were performed on 0- and 60-min samples,and the difference between the two was used to determine ureaconcentration. The urea concentration of unknown samples wascalculated from a standard curve plotted from values obtained forknown concentrations of urea hydrolyzed by urease.

Determination of Protein Content. Soluble protein content ofthe cell extract was determined by the method ofLowry et al. (16).Protein in a 0.5-ml aliquot of the cell extract was precipitated with

10% (w/v) trichloroacetic acid, heated at 100 C for 5 min andsedimented. The precipitate was washed twice with ice-cold 95%ethanol, dried, dissolved in I N NaOH, and assayed for protein.BSA dissolved in I N NaOH was used as a standard.

Cloning. The XD cells have a marked tendency to grow asuniseriate filaments of cells. Each filament consists of the progenyof a single cell. This is especially evident in exponential phase,when the filaments have the appearance of tubes with orthogonalcross walls. Therefore, clones were obtained by culturing singlefilaments isolated at the exponential phase of growth. Each fila-ment was grown on agar medium until sizable callus tissue wasformed and then transferred to a liquid medium. Single filamentswere isolated by the following methods: a portion of the culturewas poured into a sterile Petri dish and the smallest clumps visiblewith the naked eye were collected one at a time with 0.5-mlpipettes by capillary action; or single filaments were discernedwith the aid of a binocular dissecting microscope and collectedone at a time by pipetting. The first method resulted in theisolation of filaments which were each composed ofapproximately50 cells or more. With the second method, filaments composed of10 cells or less could be easily isolated. The second method wasused in the later, critical work. However, it was discovered thatfilaments consisting of 15-30 cells or more had the best chance forsurvival (plating efficiency was sometimes as high as 80%7o) whereasfilaments of less than 15 cells usually would not grow. Aftertransfer to a 0.8% agar medium (nitrate M-1D or urea M-1D), theclones took from 4 to 8 weeks to grow to callus tissue with adiameter of approximately 10 mm. The callus was then transferredinto 5-40 ml of liquid medium and allowed to grow under theculture conditions previously described. A liquid suspension soondeveloped and the cultures were maintained in the routine fashion.Urease assays were performed on the cloned cultures no soonerthan after the 3rd transfer on liquid medium.

RESULTS

Activity of Urease in Cells Grown for Many Generations onNitrate or Urea. Growth of the XD cells on urea is slower thangrowth on nitrate. The doubling time for fresh weight of the XDcells, which had been grown previously on the same nitrogensource for at least 40 generations was 2.5 days for growth onnitrate and 3 days for growth on urea. The cells grown on nitratehad specific activities of urease ranging from 0.25 to 0.55 ,imolNH4+ formed h-1 g-1 fresh weight during the culture period, whilecells grown on urea (these cells were continuously maintained onurea for 5 months prior to the experiment) had specific activitiesranging from 1.4 to 2.7 (Fig. 1). At all the times tested during theculture period, cells grown on urea had higher activities of ureasethan did cells grown on nitrate. The highest activity on bothnitrogen sources was detected during the exponential phase ofgrowth. The urease activities did not decrease to zero during thestationary phase of growth. In other experiments, cells assayedbetween 0 and 4 days did not have activities of urease higher thanthat found on the 4th day, i.e. there was not an initial jump inurease activity at the time when cells were transferred to freshmedia. When the activities of urease are expressed as ,umol NH4'formed h-' mg-' soluble protein, the cells grown on urea still haveactivities which are four to five times higher than those of cellsgrown on nitrate.The apparent variation in urease activity with culture age

cannot be attributed to lack of reproducibility of cell extraction.When one culture each of cells grown on nitrate or urea was

separated into three portions of 1 g each, extracted and assayedfor urease, the activities detected in the three separate extractsfrom cells grown on either nitrogen source did not vary more than±0.15%.A possible explanation for the different urease activities in cells

grown on nitrate or urea is that an inhibitor or activator of urease

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SLOW CHANGES IN UREASE LEVELS

00-%

-CL:_

N..

la0

Eo,0U-

I

z(n0

E

0

cn0

4 8 12

Culture Age( Days)FIG. 1. Activities of urease extracted from cells grown on urea (0) or

nitrate (0). Stationary phase cells which were previously maintained on

nitrate or urea for 5 months were subcultured into fresh medium containingthe same nitrogen source.

Table I. Urease Activities of Mixtures of Extractfrom Cells Grown on

Nitrate and Extractfrom Cells Grown on Urea

Cells grown on nitrate or urea were extracted separately. Urease activitywas determined for the same volume of each extract and for a 1:1 mixtureof half-volumes of each extract. Included is the value which would beexpected if the activities from the extracts from cells grown on nitrate or

urea were additive, i.e. if neither extract affected the activity of the other.

ExperimentUrease Activity

No.Nitrate Urea Mixed Calculated

,unol NH4' h-' g' fresh wt

1 0.31 1.64 1.13 0.972 0.54 2.12 1.38 1.33

may be present in the extract of these cells. In two experiments inwhich extract from cells grown on urea was mixed with extractfrom cells grown on nitrate, the activities of urease in the twoextracts were approximately additive (Table I). The 4- to 5-folddifference in urease activity, therefore, cannot be attributed to thepresence of an activator or inhibitor in either extract.The rate of formation of ammonium catalyzed by urease was

constant with time for at least 3 h and proportional to enzymeconcentration for extracts from both urea-grown and nitrate-grown cells. The dependence of the rate of ammonium formationupon urea concentration followed Michaelis-Menten kinetics forenzyme from cells grown on either nitrogen source, and an appar-ent Km for urea of 0.20 mM was found for the enzyme obtainedfrom either type of cells. This value is lower than the Km measuredfor urease from other plant sources (21).The differences in the activities of urease between cells grown

on nitrate or urea were consistently observed when monitoredover a time span ofapproximately I year (Fig. 2). During the year,

Sx. 2.0

1.5N.0

Z 1.061

E

@ 0.5

5 10 15 20

TronsferFIG. 2. Differences in the activities of urease in cultures grown on

nitrate or urea over the period of a year. Cultures maintained on urea

(0) or nitrate (0) were assayed for urease at various transfers over theperiod of a year. Each point on the graph represents either an activitydetermined during the exponential phase of growth or an average activitycalculated from values obtained at different ages of the culture. Thetransfer periods were 12-14 days for the cultures grown on nitrate and 16-

18 days for the cultures grown on urea.

2.5

, 2.0a

I

z

o 1.0E

0.5a

5 10 14Culture Age (doys)

FIG. 3. Urease activity in extracts of cells transferred from nitrate to

urea medium. Cells in the stationary phase of growth on nitrate were

transferred to a medium containing urea (0) or nitrate (E) as the solesource of nitrogen, and assayed for urease at the indicated times.

the urease levels of the cells grown on nitrate varied little from thespecific activity of 0.5. The urease levels in the cells grown on urea

varied between three and five times the levels in cells grown on

nitrate during this period; they never decreased to levels whichwere equal to those found in the cultures grown on nitrate.

Urease Activity of Cells Transferred from Nitrate Medium toUrea Medium. When cells which had previously grown on nitratewere transferred to urea medium and were assayed for urease atvarious times, no increase in urease was observed during theculture period (Fig. 3). The specific activities of urease from thecells growing on urea for the first time never exceeded 0.5, i.e. theactivities of urease were never higher than is normally found incells grown in nitrate. When the XD cells were transferred directlyfrom nitrate medium, they grew on urea, but the rate of growthand the yield in fresh weight at the end of the growth period were

approximately one-halfofnormal values for cells grown on nitrate.The growth rate and levels of urease in cells newly transferredfrom nitrate to urea were also consistently lower than in cells

which had grown on urea through many transfers. This phenom-enon was not due to a lack of urea uptake. The urea concentrationwas about 2.5-fold higher in the cells which were newly transferred

00

0 000

0-0

0

U.0

a a

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SKOKUT AND FILNER

from nitrate to urea, compared to cells which had grown on ureafor more than 1 year (Fig. 4). The higher concentration of urea incells newly transferred to urea may indicate that their lower levelsof urease, compared to cells which had grown on urea throughmany transfers, are insufficient to keep the cellular concentrationof urea low by hydrolysis.When cells previously grown on nitrate were subcultured on

urea medium the cells grew, but somewhat less rapidly than onnitrate medium. The level of urease activity remained low formany (about 7) transfers on urea medium. However, when thesecells were cultured on urea medium for even more transfers, theactivities of urease eventually increased 4- to 5-fold (Fig. 5). Theurease levels remained the same for the first 8 transfers, close to0.5 ,Lmol NH4' formed h-' g-' fresh wt, in the cells growing on

nitrate or urea. After the 8th transfer, the urease level of the cellsgrown on urea increased gradually to a specific activity of 2.0 atthe 15th transfer, whereas the cells growing on nitrate retainedtheir normal low activity. In a less detailed repetition of this long-term experiment, there was a similar increase in the level of ureasein the cells grown on urea, but the increase occurred earlier. Inboth experiments, the high levels of urease persisted after theinitial increase. The increase in urease level had associated with ita decrease in the urea concentration in the cells maintained onurea. In the second long-term experiment, the average intracellularconcentration of urea for all the cultures grown on urea whichexhibited the lower levels of urease was 5 times higher than theaverage urea concentration in the cells possessing the higher levels

-1 0

0.8

0.6

0.4

0.5 2 5 10Culture Age (doys)

FIG. 4. Urea concentration in cells continuously maintained on urea

(0), and in cells newly transferred from nitrate to urea medium (5). Cellsin the stationary phase of growth were transferred to a medium containingurea as the sole source of nitrogen. At various times during the cultureperiod the cells were extracted and the urea concentration of the cell

extract was determined.

c

z

,{I2

I

Trarfr

FIG. 5. Long-term experiment: urease activity of cells transferred fromnitrate to urea medium and maintained on urea thereafter. Cells previouslygrown on nitrate were transferred in the stationary phase of growth intourea (0) or nitrate (E) medium. These cultures were continuously main-tained on the two different media for 18 transfer periods. The cells were

assayed for urease during the exponential phase of growth. The threecurves were calculated from the equations in the Appendix, for fLH =

8 x 10-4, 8 x 10-5, and 8 x 10-6, using the growth rate constants 0.193day-' and 0.230 day-' for low and high urease cells, respectively. Thecurves are drawn through the urease levels calculated for the third gener-ation of each transfer.

of urease. Cells which developed elevated urease activity afterprolonged growth on urea will hereafter be termed urea-adaptedcells.

Urease Activity of Clones Isolated from Cultures Grown onNitrate or Urea. Clones were isolated from a population of cellsgrown on nitrate and assayed for urease to determine if a variationin urease activity existed on a cell to cell basis. The urease activitiesof 83 such clones (Fig. 6) were rather narrowly distributed aroundthe average value normally found in the uncloned population ofcells grown in nitrate (-k). The highest specific activity observedwas 0.8. None of the clones had an activity near the average valuepresent in uncloned populations of urea-adapted cells (- - -+). Theaverage specific activity of these clones of cells grown on nitratewas 0.34 and the SD was 0.154 (±45%). Since the variation inactivities appeared to approximate a normal distribution, a tableof integrals of the normal distribution curve (2) was used toestimate the percentage of the cell population expected to haveurease activities above or below a certain level. Only one in 500cells would be expected to have an activity greater than 0.8. Theprobability of a cell having a specific activity greater than 1.0 wasestimated to be 1 in 33,000. The probability of a cell having a

urease activity greater than 2.0 was estimated with the aid of atable of areas of the normal curve for large SD from the mean(15). From this table it was calculated that approximately 1 in 1

x 102 cells would be expected to have an activity of urease greaterthan 2.0.

Clones isolated from a population of urea-adapted cells alsoexhibit varied urease activities. The isolation of a large number ofclones from this population was difficult because the platingefficiency was less than 1% for filaments cultured on the ureamedium solidified with agar. The plating efficiency was improvedwhen filaments of urea-adapted cells were cultured on nitratemedium solidified with agar. Therefore, most ofthe clones isolatedfrom the urea-adapted cell population were first grown on solidnitrate medium for 6-8 weeks and then transferred to liquid urea

medium. By these procedures, 26 clones derived from a urea-adapted cell population which produced the higher levels ofureasewere obtained: five initially grown on solid urea medium, and 21initially grown on solid nitrate medium. The specific activities of

20 -

0

~ 1.lo 0 .

E

z

L. rr'

1.0 2.0 3.0

Urease (1imoles NH4 formed /hr./gr.fr.wt.)

FIG. 6. Urease activities of clones derived from populations of cellshaving low and high levels of urease. Clones were derived by culturingsingle filaments on solid medium. After the filaments grew to sizable callustissue they were transferred to liquid media and maintained in liquidculture. The clones were assayed for urease during the exponential phaseof growth. The urease activities of 83 clones derived from a population ofcells grown on nitrate as the sole source of nitrogen (-), and of 26clones derived from a population of cells previously grown on urea formany generations (---) are presented. The average values for urease

activity normally found in the entire population of cells grown continu-ously on nitrate ( -) or urea (-- -+) are indicated.

1.0 8

0 5 10 15

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SLOW CHANGES IN UREASE LEVELS

the urease in the clones varied about the average for specificactivity normally found in uncloned populations of urea-adaptedcells, but activities similar to those found in unadapted cell pop-ulations were not observed (Fig. 6). The lowest specific activitydetected in a clone was 1.60. The average specific activity of theseclones of urea-adapted cells was 2.43, and the SD was 0.647(±26%). Some clones had specific activities which were nearlydouble that found on the average in urea-adapted cells. Thedistribution of urease activity in the clones initially cultured onsolid urea medium was similar to the distribution in the clonesinitially cultured on solid nitrate medium.

Increase in Urease Activity during Growth on Urea in ClonesDerived from a Population of Cells Previously Grown on Nitrate.Eight clones were isolated from a population of cells grown onnitrate, cultured on agar-urea medium, and subsequently main-tained in liquid medium containing urea as the nitrogen source.At the exponential phase of growth of the 5th and 10th transfersthey were assayed for urease (Table II). At the 5th transfer, theclones had urease levels which were still quite close to that of theoriginal cell line. However, the urease activity increased in all ofthe clones from the 5th to the 10th transfer. In four of the clones,the urease activity at the 10th transfer was at least double theactivity at the 5th transfer. It appeared that all of the clones werein the process of adapting to urea and acquiring the higher levelsof urease. The average of the urease levels in these clones at the10th transfer was identical to that observed at the 10th transfer inthe long-term experiment with uncloned cells (Fig. 5).Comparison of Growth Rates of Cultures which Exhibit the

High or Low Levels of Urease. Urea-adapted cells grow faster onurea than do unadapted cells (Fig. 7). Growth rate constants anddoubling times have been calculated using the usual equations:

CtIn-

COk=

t

0.693= t2

k

where k = rate constant for growth, Ct = fresh weight at day 15,C. = initial fresh weight of cells, t = time (15 days), and t2 =doubling time. While it is evident (Fig. 7) that the fresh weightincrease deviated substantially from simple exponential growth,these approximations are nevertheless useful for estimatingchanges in relative abundance of cells in mixed populations. The

Table II. Urease Activity of Clones Isolatedfrom a Population of CellsGrown on Nitrate and Transferred to Urea Medium

Single filaments were isolated from a culture in the exponential phase(4 days old) of growth on nitrate and grown on agar medium containingurea as the sole source of nitrogen. After 7 weeks, the callus tissue formedwas cultured in liquid medium containing urea. The clones were assayedfor urease at the exponential phase of growth after 5th and 10th transferson liquid medium. The urease activity of the original uncloned parentculture was 0.45. The average urease activity at the 5th transfer was 0.54with a SD of 0.14 (±26%). The average urease activity at the 10th transferwas 0.98 with SD of 0.22 (±22%).

Urease ActivityClone No.

5th Transfer 10th Transfer

Ftnol NH4' h-' g' fresh wt1 0.38 0.732 0.68 0.963 0.48 0.974 0.43 0.995 0.59 0.926 0.55 1.487 0.67 0.878 0.51 0.89

*=l-J

3 6 9 12 15Culture Age (Days)

FIG. 7. Differences in growth on urea of high urease and low ureasecells. Cells in the stationary phase of growth were weighed under sterileconditions so that all inocula were identical and transferred to freshmedium containing urea as the sole source of nitrogen. At 3-day intervalsduring the growth period, triplicate samples of the cultures were harvestedand the fresh weight determined. Data are presented for urea-adaptedcells which possess higher levels of urease (0); cells recently transferredfrom nitrate medium to urea medium and maintained on urea for threetransfers, which possess lower levels of urease ([J); and cells newlytransferred from nitrate to urea which possess lower levels of urease (A).

growth rate constants and doubling times were 0.230 day-' and3.0 days for the high urease cells growing on urea, 0.193 day-'and 3.6 days for the low urease cells growing on urea and 0.136day-' and 5.1 days for the cells newly transferred from nitrate tourea. The differences in the rates ofgrowth of the high urease andlow urease cells on urea were consistently observed for the culturesused in all of the experiments reported in this paper. Since thecells grow more rapidly on nitrate than on urea (11) even withhigh urease, there exists unrealized potential for even highergrowth rates on urea.

Effect of Mixing Nitrate-grown and Urea-adapted Cells on theRise in Urease Activity. A mixing experiment was performed todetermine whether or not low urease cells and high urease cellsbehave independently in mixed populations. Stationary phasecultures of urea-adapted cells with high urease and unadaptednitrate-grown cells with low urease were transferred to urea me-dium and were mixed at ratios of 100:0, 50:50, 10:90, and 0:100.These cultures were maintained for three transfers and assayedfor urease during the exponential phase of growth in each period(Table III). Included in Table III is the estimated ratio of the twocell types in the population at the time when urease was assayed,i.e. the ratio expected if the two cell types multiplied independ-ently. The expected ratios were calculated by using the growthrate constants for the three different cell types reported in theprevious section. The growth rate constant of 0.230 day-' wasused for the urea cells whereas the growth rate constant of 0.136day-' was used for the nitrate cells of the 1st transfer period and0.193 day-' was used for the nitrate cells of the 2nd and 3rdtransfer periods. Also included in the table are the predicted valuesfor activities of urease which would be present if the culturescontained the calculated ratios of cells. In all cases, the calculatedactivities were not appreciably different from the observed activ-ities of the mixed cultures. Apparently, neither 10 nor 50%Yo highurease cells could cause the low urease cells to develop high levelsof urease.

Effect of Various Nitrogen Sources on the Activity of Urease inUrea-adapted Cells. The stability of the high urease level in urea-

Plant Physiol. Vol. 65, 1980 999

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SKOKUT AND FILNER

Table III. Urease Activity of Mixtures of Urea-adapted Cells and Nitrate-Grown Cells Cultured on Urea Medium

Stationary phase cells previously grown on nitrate (N) or urea (U) werecultured into urea medium at ratios of 100 U:0 N, 50 U:50 N, 10 U:90 N,and 0 U: 100 N. At stationary phase the cultures were transferred to freshurea medium for three transfer periods. The cultures were assayed forurease during the exponential phase of growth at each transfer period.

Cell Ratio U:Na Urease ActivitybTransfer

Initial Expected Observed Expected

ymol NH4+formed h'g 'fresh wt

1st 100:0 2.6650:50 65:35 1.64 1.9010:90 14:86 0.89 0.790:100 0.49

2nd 100:0 2.5050:50 87:13 1.90 2.2510:90 42:68 1.35 1.470:100 0.62

3rd 100:0 2.4650:50 92:8 2.00 2.2210:90 55:45 1.90 1.600:100 0.59

a Because the rates of growth on urea are different for the nitrate-grownand urea-adapted cells, the change in the ratio of nitrate to urea cellsbrought about by the difference in the growth rates was calculated for theday of assay using the growth rate constants determined from Figure 7.

b An expected activity was calculated from the activity observed in thenitrate and urea cell cultures and the calculated ratios of U cells:N cellsfor the mixed cultures in that transfer period.

adapted cells was tested by transferring these cells to media whichcontained altemate sources of nitrogen. When cells with highurease were transferred to a medium which contained nitrate asthe sole source of nitrogen and continuously grown on nitratethereafter, the urease levels of these cells at the exponential phaseof growth did not immediately decrease to the levels normallyfound in cells routinely grown in nitrate (Fig. 8a). During thegrowth period of the Ist transfer these cells were making highlevels of urease even though urea was not present in the medium.The urease activity gradually decreased in these cells during thefirst four transfers until a specific activity of 1.0 was obtained.After the 4th transfer these cells appeared to have a urease specificactivity which had stabilized around 1.0. The gradual and parallelrises observed after the 11th transfer in both cultures (Fig. 8)probably have nothing to do with the nitrogen source. For un-known reasons, about that time, cultures began producing slightlyhigher yields, suggesting a change in some uncontrollable envi-ronmental parameter, e.g. traces of growth inhibitors may nothave been present in new chemicals used to make media. This isone of the hazards associated with experiments conducted over along time span.A similar experiment was performed using ammonium succi-

nate as the nitrogen source (Fig. 8b). During the first few transferperiods, the cells grown on ammonium succinate had activities ofurease which were roughly one-half that of the activity found insimilar cells grown on urea. On the 9th transfer, the cells main-tained on ammonium succinate had a specific activity of urease of0.5. The cells continued to exhibit this low level of urease activityfor the next seven transfers. The ammonium present in extractsfrom cells grown on ammonium succinate did not appear to havea direct effect on the activity of urease for the following reasons:(a) removal by dialysis of ammonium succinate or ammoniumfrom the extracts of cells grown on ammonium or urea, respec-tively, did not result in an increase in activity of urease; (b) the

T 2 0 00

* 0~~~~~~~a~~~IOo~~~~T ~~~~~~~~0

3E b 0 o°a5. 00I ~~~0

Z 0

2.0 0°

1.0 a

5 10 15Transfer

FIG. 8. Gradual decrease in urease activity in urea-adapted cells trans-ferred to, and maintained on, nitrate medium (a) or ammonium succinatemedium (b). Urea-adapted cells in the stationary phase of growth weretransferred to urea medium (0) and to either nitrate medium (5) orammonium succinate medium (A). The cultures were maintained on thesame nitrogen source for 16 transfer periods. The cultures were assayedfor urease at the exponential phase of growth for most of the transferperiods.

'_ 2.0 /

x. -

0-In 1.5 /

z 1.0 _z

E 0.5

2 4 6Transfer

FIG. 9. Increase in urease activity when cells previously grown on

ammonium succinate are transferred back to urea medium. Cells previ-ously grown on ammonium succinate (from experiment of Fig. 8b) were

transferred to urea (0) or ammonium succinate (5) medium and main-tained on these media for five transfer periods. At the exponential phaseof growth, the cultures were assayed for urease.

ammonium concentrations of extracts from the cells grown onurea (determined from the zero time point of the urease assay)were equal to or slightly higher than the ammonium concentra-tions of extracts from the cells grown on ammonium succinate;and (c) the urea concentration used for the urease assay wasapproximately 100-fold higher than the ammonium concentrationin the cell extracts. When the cells grown on ammonium succinate,which were now making the low levels of urease, were transferredback to urea medium, it took only three transfer periods beforethey were again making the higher levels of urease (Fig. 9). This

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SLOW CHANGES IN UREASE LEVELS

increase in urease which began during the first transfer period inurea medium was considerably faster than the increase detectedin cells that had never been exposed to urea when they weretransferred from nitrate to urea for the first time (Fig. 5).Casamino acids were also tested for their effect on the levels of

urease in urea-adapted cells. When cells were continuously cul-tured on casamino acids the urease levels decreased quickly; atthe exponential phase of the 4th transfer period these cells had aspecific activity of 0.47. The casamino acids present in the cellextracts did not inhibit the activity of urease because dialysis ofthe extract from cells grown on casamino acids did not increaseactivity, and mixing of extracts from cells grown on urea and cellsgrown on casamino acids did not inhibit activity.

DISCUSSION

The object of this investigation was to characterize the regula-tion of urease in the tobacco XD cells. The apparent induction ofnitrate reductase (7) and of nitrite reductase (14) by nitrate ornitrite, and the apparent derepression of acid phosphatase by theabsence of phosphate (27) and of ATP sulfurylase by the absenceof sulfur (22) have been documented in the XD cell line. It wasanticipated that urea might similarly induce urease in these cellsbecause of the previous reports of apparent induction of urease inother higher plant tissues (5, 17, 18, 20), and because it can serveas an optional nitrogen source for growth of XD cells (4, 1 1) andother plant tissue cultures (4, 12, 20, 29).

Urease in the tobacco XD cells is indeed responsive to urea inthe culture medium. This became evident from the finding thatcells grown continuously on urea for many generations haveactivities of urease which are 4 or 5 times higher than those foundin the cells grown on nitrate (Fig. 1). The properties of ureaseextracted from cells grown on the two different nitrogen sourcesare similar; for instance, they possess exactly the same Km for urea.However, the proteins responsible for the high and low ureaseactivities must be characterized in detail before a definitive state-ment can be made concerning their identities.The urea-urease system differs from the nitrate-nitrate reductase

system in these cells in several ways. Urease is present at a lowconstitutive level in XD cells not exposed to urea, whereas nitratereductase normally is formed only if an inducer is present. TheXD cells do not respond to their initial exposure to urea byimmediately making higher levels of urease (Fig. 3), but theyrespond to nitrate by immediately making nitrate reductase (30).Furthermore, cells which possess the higher levels of urease retainhigh levels through a few transfers into medium without urea (Fig.8). Nitrate reductase on the other hand begins to decay within afew hours after the inducer is removed. The mechanism determin-ing the changes in the levels of urease in these cells appears to befundamentally different from that responsible for the much fasterchanges associated with nitrate reductase and other regulatedenzymes in these cells.At least two kinds of mechanisms might generate a slow increase

in urease activity in cells which have been transferred from nitrateto urea medium. These are: (a) selection mechanisms; a sponta-neous mutant or stable (or metastable) variant with high ureasecould arise independently of the nitrogen source. The high ureaseactivity could enable the progeny of the mutant/variant to growmore rapidly on urea than the low urease population, and there-fore eventually become the dominant cell type in the culture; and(b) induction mechanisms; urea could slowly cause some or all ofthe cells to accumulate higher amounts of urease. A third possi-bility is a hybrid mechanism in which urea would induce highurease in a few cells, and the more rapidly growing progeny ofthose cells would become dominant in the culture.One prerequisite of a selection mechanism is that the high

urease cells must have a selective advantage. The XD line of

tobacco cells grows less rapidly on urea as sole source of nitrogenthan it does on the same medium containing nitrate instead ofurea. During growth on urea, the highest levels of intracellularurea accumulate in cells with the lowest urease activity (Fig. 4),and when the urease level eventually increases in these cells, thegrowth rate increases, and the urea concentration decreases. It canbe inferred from these observations that: (a) growth rate on urea

is nitrogen-limited; (b) urease is responsible for urea utilization inthese cells; and (c) the rise in urease level is responsible for thedecrease in accumulation of urea, hence the increase in thenitrogen-limited growth rate. Therefore, the slightly higher rateconstant for growth on urea nitrogen of the high urease cells(0.230 day-) compared to that of low urease cells (0.193 day-l)should provide a selective advantage. Evidence in support of thiscomes from the finding that urease levels observed in mixedpopulations of high urease cells and low urease cells were in goodagreement with those expected on the assumption of independentmultiplication of the two populations (Table III).One can also ask if the observed rise of urease activity during

the slow adaptation to urea fits with that predicted from the rateconstants for multiplication of high and low urease cells. Whencalculations were done (see Appendix) for a number of differentmodels, it became apparent that the shape of the rise curve was

determined by the two rate constants, but the time of onset of therise was determined by the manner and frequency of originationof high urease cells. The observed curve (Fig. 5) could be generatedby assuming that high urease cells arose from low urease cells atmitosis at a frequency of 8 x 1-0, then multiplied at theircharacteristic rate. The curve could also be generated by assumingthat only during the initial brief transition from growth on nitrateto growth on urea did high urease cells arise and that they did so

at a frequency of 2 x I0` after which they multiplied at theircharacteristic rate. Still a third possible model is one in which theconversion of a low urease cell to a high urease cell has a constantprobability at all times, rather than being non-zero only at theinitial transition from nitrate to urea, or at mitosis. For this model,the probability would be 8 x 1-0 per 3.6 days, the generationtime of low urease cells.The good fit of the observed dependence of urease level on

number of transfers on urea medium, with that predicted forselection models, does not prove the correctness of the selectionhypothesis. However, it is strong supporting evidence. Only one

curve shape can be predicted by simple selection hypotheses, whileinduction hypotheses place no such constraint on the curve.

The most intriguing question about slow adaptation to urea is:what is the origin of the high urease cells? One possibility is thatthey are always present as a result of heritable random variationof urease levels in the cell population but require growth on urea

to have a selective advantage. Such variation could conceivablyarise, for example, as a result of nonspecific mutations mildlyaffecting some aspect of protein synthesis or degradation. Basedon the distribution of low urease activities in 83 clones isolatedfrom a cell population having low urease (Fig. 6), the probabilityof a clone having a high urease level of 2 ,umol NH4+ formed h-'g 1 fresh wt was estimated to be about 10-23. Since cultures were

grown to about 4.5 x 107 cells per culture, the probability ofencountering a high urease clone descended from a cell that arose

by the random variation of urease level which generated theobserved distribution is negligible. High urease cells must arise atfar higher frequency, hence by another mechanism of variation,to account for the observations.A possibility which must be considered is that the high urease

variants arise by the same kind of mechanism which has generatedother biochemical variants of the tobacco XD cells. Variantsresistant to L-threonine (11), selenoamino acids (8) and otheramino acid analogs (28) have been isolated and their frequencieshave been estimated to be about l0_. If high urease cells arose at

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1002 SKOKUT A

that low frequency, about 24 transfers would have been requiredbefore the high urease cells would have dominated sufficiently toelevate the urease level according to calculations based on theselection model. The only possible way that a mechanism whichgenerates variants at a frequency of l0-7 could account for therise in urease is if the variants existed before the shift to ureamedium, at a frequency of about 2 x 10-3, as a consequence ofsome fortuitous prior selection. However, evidence that highurease cells do not have to pre-exist in a low urease cell populationwas provided by following the urease levels in 8 clones of lowurease cells transferred to urea medium. In all cases, the ureaselevel had risen by the 10th transfer, indicating that high ureasecells had originated from low urease cells.

Based on the above considerations, it appears most likely thathigh urease cells originate from low urease cells at a frequency ofabout 8 x 10-5 per generation and then gradually overgrow thelow urease cells. This frequency is reminiscent of some of theperplexingly high frequencies (around l0-4) reported for culturedanimal cell variants selected for resistance to anti-metabolites (10).Because of their high frequency, instability and noninducibilityby mutagens, it is doubted that such animal cell variants aremutants.High urease variants of tobacco XD cells are not stable when

the cells are grown on nitrogen sources other than urea. A threo-nine-resistant variant isolated from the XD cell line persistedthrough many transfers in the absence of the selective agent (I 1),but the high urease level of urea-adapted cells declined afterseveral transfers in media containing nitrate (Fig. 8a), ammoniumsuccinate (Fig. 8b) or casamino acids. The gradual decrease inurease activity during growth on nitrate was different from thatduring growth on other nitrogen sources because the activity didnot decrease to the original low levels even after 18 transfers.Another oddity of the response of the XD cells to urea was seen

in the response of the cells to a second period of growth on urea.When cells which had achieved the high urease level after pro-longed growth on urea, and then the low urease level duringgrowth on ammonium succinate, were again transferred to urea,the urease level rose within two transfers (Fig. 9), a much morerapid rise than occurred during the first series of transfers on urea.The rise occurred too rapidly to be attributable to reselection ofhigh urease cells. The cells somehow had "remembered" that theirancestors had at one time grown on urea.The alternative to the selection hypothesis is the specific induc-

tion by urea hypothesis. We do not at present have evidence foror against this alternative. To discriminate between these twohypotheses, it would be necessary to determine if the urease levelrises in a single sharp step in rare cells, or in a continuum ofincrements in all cells. This could perhaps be done best bydetermining the distribution of urease levels in clones isolatedfrom a population in which the urease activity is just beginning torise. The selection hypothesis predicts a discontinuous bimodaldistribution with maxima at the high and low urease levels, whilethe induction hypothesis predicts a unimodal distribution with amaximum intermediate between the two extremes.Although the molecular mechanisms responsible for the in-

crease in urease reported here are not yet established, the similar-ities of this phenomenon to the increases observed in dihydrofolatereductase levels in murine Sarcoma cells (1) suggest that duplica-tion of the gene for urease may be a possibility. In both systemsthe changes involve the level of a specific constitutive enzymewhich appears to be growth-limiting. The changes occur gradually,and once the change has occurred the new levels remain stable ifthe cells are kept under the same culture conditions. When theculture conditions are changed (omission of methotrexate fromthe sarcoma cell culture medium or omission of urea from thetobacco cell culture medium, Fig. 8), the enzyme levels are unsta-ble and gradually decrease to the original lower levels. In some

ID FILNER Plant Physiol. Vol. 65, 1980

cases, the levels of dihydrofolate reductase stabilize at an elevatedlevel in the absence of methotrexate (19). In the tobacco cells theurease levels are stabilized at a point between the high and lowlevels when the cells are transferred back to nitrate medium (Fig.8a). However, the increases in dihydrofolate reductase occur as aresult of a progressively more stringent stepwise selection proce-dure in which the cells are exposed to increasingly higher concen-trations of methotrexate and most of the cells are killed at eachconcentration step. The increase in urease does not involve adrastic selection procedure. The increase observed in dihydrofolatereductase activity can be 200-fold higher than the normal levels,and dihydrofolate reductase can comprise as much as 6% of thetotal soluble protein (1). The highest increase observed in ureaselevels was 7-fold. If the increase of urease level does reflect genedosage, increases of the observed magnitude could result frompolyteny, polysomy or polyploidy, rather than multiplication ofspecific genes within a chromosome. The high concentration ofurease in the seeds of some plants (3) raises the intriguing possi-bility that if selection for multiple copies of the urease gene hasoccurred in the laboratory, it may have occurred earlier in nature.

LITERATURE CITED

1. ALT FW, RE KELLEMS, JR BERTINO, RT SCHIMKE 1978 Selective multiplicationof dihydrofolate reductase genes in methotrexate-resistant variants of culturedmurine cells. J Biol Chem 253: 1357-1370

2. ARKIN H, RR COLTON 1970 Tables for Statisticians. Barnes & Noble, Inc, NewYork, p 119

3. BAILEY CJ, D BOULTER 1971 Urease, a typical seed protein of the Leguminosae.In JB Harborne, D Boulter, BL Turner, eds, Chemotaxonomy of the Legumi-nosae. Academic Press, New York, pp 485-502

4. BEHREND J, RI MATELES 1975 Nitrogen metabolism in plant cell suspensioncultures I. Effect of amino acids on growth. Plant Physiol 56: 584-589

5. BOLLARD EG, AR COOK, NA TURNER 1968 Urea as sole source of nitrogen forplant growth I. The development of urease activity in Spirodela oligorrhiza.Planta 83: 1-2

6. FILNER P 1965 Semi-conservative replication of DNA in a higher plant cell. ExpCell Res 39: 33-39

7. FILNER P 1966 Regulation of nitrate reductase in cultured tobacco cells. BiochimBiophys Acta 118: 299-3 10

8. FLASHMAN SM, P FILNER 1978 Selection of tobacco cell lines resistant toselenoamino acids. Plant Sci Lett 13: 219-229

9. GRANICK S 1937 Urease distribution in plants. General methods. Plant Physiol12: 471-486

10. HARRIS M 1964 Cell Culture and Somatic Variation. Holt, Rinehart and Winston,New York, pp 337-341

11. HEIMER YM, P FILNER 1970 Regulation of the nitrate assimilation pathway ofcultured tobacco cells II. Properties of a variant cell line. Biochim BiophysActa 215: 152-165

12. JONES RW, AJ ABBoTT, EJ HEwITT, DM JAMES, GR BEST 1976 Nitrate reductaseactivity and growth in Paul's Scarlet Rose suspension cultures in relation tonitrogen source and molybdenum. Planta 133: 27-34

13. KAPLAN A 1965 Urea nitrogen and urinary ammonia. In S. Meites ed, StandardMethods of Clinical Chemistry, Vol 5. Academic Press, London, pp 245-256

14. KELKER HC, P FILNER 1971 Regulation of nitrite reductase and its relationshipto the regulation of nitrate reductase in cultured tobacco cells. Biochim BiophysActa 252: 69-82

15. LOWAN AN 1942 Tables of probability functions, Work Projects Administrationfor the City of New York, Vol 2. Sponsored by the National Bureau ofStandards, p 340

16. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-ment with the Folin phenol reagent. J Biol Chem 193: 265-275

17. MATSUMOTO H, T YASUDA, M KOBAYASHI, E TAKAHASHI 1966 The inducibleformation of urease in rice plants. Soil Sci Plant Nutr 12: 33-38

18. MOKRONosov AT, ZG ILINYKH, NI SHUKOLYUKOVA 1966 Assimilation of ureaby potato plants. Fiziol Rast Mosc 13: 707-713

19. NAKAMURA H, JW LITTLEFIELD 1972 Purification, properties, and synthesis ofdihydrofolate reductase from wild type and methotrexate-resistant hamstercells. J Biol Chem 247: 179-187

20. POLACCO JC 1976 Nitrogen metabolism in soy bean tissue culture I. Assimilationof urea. Plant Physiol 58: 350-357

21. REITHEL FJ 1971 Ureases. In PD Boyer, ed, The Enzymes, Ed 3, Vol 4. AcademicPress, New York, pp 1-21

22. REUVENY Z, P FILNER 1975 Regulation of adenosine triphosphate sulfurylase incultured tobacco cells. J Biol Chem 252:1858-1864

23. ROON RJ, B LEVENBERG 1970 CO2 fixation and the involvement of allophanatein the biotin-enzyme-catalyzed cleavage of urea. J Biol Chem 245: 4593-4595

24. ROON RJ, B LEVENBERG 1972 Urea amidolyase. I. Properties of the enzyme fromCandida utilis. J Biol Chem 247: 4107-4113

LN

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SLOW CHANGES IN UREASE LEVELS

25. SKOKUT TA, P FILNER 1977 Slow adaptation of urease levels in tobacco cellscultured on different nitrogen sources. Plant Physiol 59: S-10

26. THOMPSON JF, AE MUENSTER 1974 ATP-dependent urease: characterization ofand control in Chlorella: The search for it in higher plants. In RL Bieleski, ARFerguson, MM Cresswell, eds, Mechanisms of Regulation of Plant Growth,Bulletin 12. Royal Society of New Zealand, Wellington, pp 91-97

27. UEKI K, S SATO 1971 Effect of inorganic phosphate on the extracellular acidphosphatase activity of tobacco cells cultured in vitro. Physiol Plant 24: 506-511

28. WIDHOLM JM 1976 Selection and characterization of cultured carrot and tobaccocells resistant to lysine, methionine and proline analogs. Can J Bot 54: 1523-1529

29. YOUNG M 1973 Studies on the growth in culture of plant cells XVI. Nitrogenassimilation during nitrogen-limited growth of Acer pseudoplatanus L. cells inchemostat culture. J Exp Bot 24: 1172-1185

30. ZIELKE HR, P FILNER 1971 Synthesis and turnover of nitrate reductase inducedby nitrate in cultured tobacco cells. J Biol Chem 243: 1772-1779

APPENDIX

Calculation of expected urease levels, assuming high ureasecells arise at mitosis of low urease cells at a constant frequency,and multiply on urea more rapidly than low urease cells.

n = number of low urease cell generationsLn = population of low urease cells after n generationsHn = population of high urease cells after n generationst2L = generation time of L cellskH = exponential rate constant for H cells

1003

fLH = frequency of origination of H cells at mitosis of L cellsUn= urease specific activity of the mixed population after n

generations of growth on urea.

Generation L cellsNo.

H cells

0 L

1 22LC- fLH2 Lo

2 4 Lo fLH4Lo+ fLH2Lo ekH2LI

4\3 8 Lo- f 8 L H+fL4 L ekH + fLH 2 Lo ekH2L2

n L.= 2nLon-l

Hn = fLH Lo E 2n-aekHt2La-O

Assume that the urease specific activity of L cells is 0.5, and theurease specific activity of H cells is 2.0.

Un = 0.5L

+ 2.0 (.

)

This equation was used to calculate the curves in Figure 5, forthree values of fLH.

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